Cybrids - Revision history

Raj at 08:00, 18 June 2009

2009-06-18T08:00:45Z

@@ Line 1: / Line 1: @@
 =Project=
 =Strategy=
-* Requierements:
+==Protocol==
 ** Inbred lines of N glauca and N.langsdorffii-
 ** Sowing: glass house
 * Protoplast isolation:
-** 1. Young leaves,----surface sterilize with 70% alcohol, leaf cut into strips-- placed in an enzyme solution containing 1:1 mixture of M2 culture medium and E1 enzyme solution (2% cullulase, 2% Hemicellulase Rhozyme and 1% pectinase) incubate for 4hrs @ lab conditions (23degree)--- initial obtained protoplast will be inferion in quality so discard----and add Enzyme M2 medium and incubate for 30min to the remaining leaf fragments.
+** 1. surface sterilize young leaves with 70% alcohol, cut leaf into trips-- placed in an enzyme solution containing 1:1 mixture of M2 culture medium and E1 enzyme solution (2% cullulase, 2% Hemicellulase Rhozyme and 1% pectinase) incubate for 4hrs @ lab conditions (23degree)--- initial obtained protoplast will be inferion in quality so discard----and add Enzyme M2 medium and incubate for 30min to the remaining leaf fragments.
 --newly released protoplast are mixed (Both male and female) and filter through 80um stainless steel mesh or miracloth to a centrifuge tube---- wash that in solution D (3.5 mM CaCl2, 0.7 mM KH2PO4, 0.5M glucose and 1lt of dist. water)
@@ Line 43: / Line 85: @@
 ---% viable pollen was counted in mounts prepared with lactophenol aniline blue,
 ---all parasexual hybrids were selfed to obtain progeny.
 =Resources=

Raj at 16:49, 15 June 2009

2009-06-15T16:49:58Z

← Older revision		Revision as of 16:49, 15 June 2009
Line 1:		Line 1:
	==
	=Project=		=Project=

	=Strategy=		=Strategy=


	* Requierements:		* Requierements:
	** Inbred lines of N glauca and N.langsdorffii-		** Inbred lines of N glauca and N.langsdorffii-

Raj: New page: == =Project= =Strategy= * Requierements: Inbred lines of N glauca and N.langsdorffii- Sowing: glass house * Protoplast isolation: ** 1. Young leaves,----surface sterilize with 70% ...

2009-06-15T16:46:59Z

New page: == =Project= =Strategy= * Requierements: ** Inbred lines of N glauca and N.langsdorffii- ** Sowing: glass house * Protoplast isolation: ** 1. Young leaves,----surface sterilize with 70% ...

New page

==
=Project=
=Strategy=

* Requierements:
** Inbred lines of N glauca and N.langsdorffii-
** Sowing: glass house
* Protoplast isolation:
** 1. Young leaves,----surface sterilize with 70% alcohol, leaf cut into strips-- placed in an enzyme solution containing 1:1 mixture of M2 culture medium and E1 enzyme solution (2% cullulase, 2% Hemicellulase Rhozyme and 1% pectinase) incubate for 4hrs @ lab conditions (23degree)--- initial obtained protoplast will be inferion in quality so discard----and add Enzyme M2 medium and incubate for 30min to the remaining leaf fragments.
--newly released protoplast are mixed (Both male and female) and filter through 80um stainless steel mesh or miracloth to a centrifuge tube---- wash that in solution D (3.5 mM CaCl2, 0.7 mM KH2PO4, 0.5M glucose and 1lt of dist. water)

-- pipette 150ul of protoplast suspension on to a 22x22mm coverslip, place this in petri dish to reduce evaporation.
-- A tiny drop of silicone 200 fluid 100cs wil be used to hold the cover slip in position. when protoplast settled onto the cover slip,

--add 450 ul of PEG solution of P2 (PEG 0.33M (1540), CaCl2.2H2O-10.5 mM, Kh2(PO4).H2O-0.7mM in a lt of water (pH 5.5, Molality 0.34)) to the protoplast culture.

--incubate at room temp and ordinary light conditions in labfor 30 mins.

-- Adhesion of membranes began immediately.

--add 500ul of M3 medium, incubate for 10 mins, and wash the protoplast 5 times with 10ml of M3 medium.
-- culture containing fusion products were maintained with low light (50-100 lux) warm temp with minimum vibration.

-- after few days, culture medium being used up and colonies began to grow,additional drops of M3 medium added in to suspension

-- when visible colonies were formed (2-3mm dia), transfer it to agar medium containing sucrose, mineral salt and iron as in MS formulation, vitamins of B5 medium and no phytohormones in 60ml specimen container.

--- Cultures were maintained under 26 degree temp, light (1000lux) for 16 hrs and 22degree in the dark for 8 hrs until calli formed as peanut size.

---Protoplast preparation, fused and suspended in M3 medium---15 days
---Calli sub culturing on hormoneless media-------------------------20days--- (8/culture)
---differentiation, analyse cytologically---------------------------------7 weeks after fusion
----second sub culturing------------------------------------------------------10weeks after fusion

---all subsequent subcultures were grown on MS media containing Major and minor salts, 30 g/l sucrose, 27.8 mg/l iron and no vitamins, glycine,inositol or hormones.
--- Medium was solidified with Bacto-agar at 0.8% with a pH of 5.5.
----sub cultures were grown at a 25 degree temp, 16 hrs of light at 5000 lux ot 8hrs dark.
----12 weeks after fusion, some cultures had differentiated as leaf and stem (no roots)
--- so they grafted on to the young plants of N. langsdorffii or N.gluaca.
---- 4 months after fusion first roots were produced on calli. these could then be transplanted into soil in pots ingreen house after initial few weeks of intensive care in mist section.
----7 months after fusion, first para sexual hybrid began to flower.
---- somatic chromosome no. were determined from acetocarmine smears of bud stage corollas,
pairing observed in metaphase-I, in pollen mother cells.
---% viable pollen was counted in mounts prepared with lactophenol aniline blue,
---all parasexual hybrids were selfed to obtain progeny.

=Resources=

Cybrids - Revision history

Raj at 08:00, 18 June 2009

Raj at 16:49, 15 June 2009

Raj: New page: == =Project= =Strategy= * Requierements: ** Inbred lines of N glauca and N.langsdorffii- ** Sowing: glass house * Protoplast isolation: ** 1. Young leaves,----surface sterilize with 70% ...

Raj: New page: == =Project= =Strategy= * Requierements: Inbred lines of N glauca and N.langsdorffii- Sowing: glass house * Protoplast isolation: ** 1. Young leaves,----surface sterilize with 70% ...